Information about CD1-lipid complex formation comes mainly from antigen presentation assays and crystal structures in which the experimentalist adds a specific lipid of interest.  To measure the spectrum of endogenous self lipids captured by cellular CD1 proteins, we developed a high performance liquid chromatography-time of flight mass spectrometry-based lipidomics platform.  This platform separated and enumerated lipids with low rates of false positive ligand detection.  Human CD1b proteins bound hundreds of ligands that spanned a broad range of polarity, including hydrophobes, glycolipids, phospholipids, phosphoglycolipids and zwitterionic lipids.  After solving the structures of lipid types that bind multiple CD1 proteins, we used these promiscuously binding lipids as tools to probe the functional size capacity of CD1 clefts and matched these to the volumes of the hydrophobic clefts observed in crystal structures. Unlike control proteins, CD1b ligands showed a mismatch in lipid anchor length as compared to the volume of the hydrophobic cleft.  These functional and structural data support a two-chamber model for lipid capture by cellular CD1b proteins.   Deeply buried scaffold lipids and protruding antigenic ligands simultaneously bind newly translated CD1b proteins.  Based on exchange of one or two cleft blocking lipids, this mechanism provides a solution to the previously unexplained phenomenon in which small exogenous lipids readily load onto CD1b, whereas loading of large bacterial lipids require lysosomal recycling.